Compounds and Compositions for Ossification and Methods Related Thereto

ABSTRACT

The disclosure relates to compounds and compositions for forming bone and methods related thereto. In one embodiment, the disclosure relates to a composition comprising a compound disclosed herein, such as 2,4-diamino-1,3,5-triazine derivatives or salts thereof, for use in bone growth processes. In a typical embodiment, a bone graft composition is implanted in a subject at a site of desired bone growth or enhancement.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/334,030 filed Oct. 25, 2016, which is a division of U.S. applicationSer. No. 13/816,312 filed Feb. 11, 2013, which is the National StageApplication of International Application No. PCT/US2011/048252 filedAug. 18, 2011, which claims priority to U.S. Provisional Application No.61/374,667 filed Aug. 18, 2010 and U.S. Provisional Application No.61/479,910 filed Apr. 28, 2011. The entirety of each of theseapplications is hereby incorporated by reference for all purposes.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 10164USCON ST25.txt. The text file is 10 KB, wascreated on Nov. 2, 2017, and is being submitted electronically viaEFS-Web.

FIELD

The disclosure relates to compounds and compositions for forming boneand methods related thereto. In one embodiment, the disclosure relatesto a composition comprising a compound disclosed herein, such as2,4-diamino-1,3,5-triazine derivatives or salts thereof, for use in bonegrowth processes. In a typical embodiment, a bone graft composition isimplanted in a subject at a site of desired bone growth or enhancement.

BACKGROUND

Bone grafting is typically performed for spinal fusions, after cancerousbone removal, and in certain operations, e.g., plastic surgery. Theiliac crest is often used as a donor site for autologous grafts.Complications collecting bone from the iliac crest include pain, nervedamage, hematoma and wound complications, avulsion of the anteriorsuperior iliac spine (ASIS), hematoma, herniation of the abdominalcavity contents, and cosmetic deformity. Thus, it is desirable todevelop materials and methods of forming bone that do not requireharvesting bone from remote sites of the patient.

Synthetic bone grafts typically include a matrix that holds minerals andother salts. Natural bone has an intracellular matrix mainly composed oftype I collagen, and some synthetic bone grafts include a collagenmatrix. Synthetic bone grafts typically contain bone growths factorssuch as bone morphogenetic proteins (BMPs) because of their ability toinduce ossification in the matrix material. Recombinant human BMP-2 hasbeen approved by the FDA in synthetic bone grafts such as INFUSE.INFUSE™ is approved for open tibial shaft fractures, lumbar interbodyfusion, and sinus and alveolar ridge augmentations. However, the highcost and need for high concentrations of BMP-2 for treatment creates abarrier for routine clinical use. Thus, there is a need to identifyadditional compositions that can substitute or complement the use ofBMPs in treating bone-related conditions.

Cellular response to BMPs depends on a complex set of interactionstypically involving intracellular signaling proteins known as Smads. Thebaseline levels of Smads are in part affected by their ability tointeract with Smurf1, a key regulator of the degradation of BMP-2signaling molecules, Smad1 and Smad5. Smurf1 interacts with Smad1/5 andtargets them for degradation, thus, leading to reduced BMP signaling.

SUMMARY

The disclosure relates to compounds and compositions for ossificationand methods related thereto. In certain embodiments, it is an object ofthe disclosure to provide certain compounds, compositions, and methodsof using compounds to improve bone grafting or to induce bone formationin a subject. In typical embodiment, the bone graft compositioncomprises a compound that modulates Smurf1 interaction with its naturaltarget proteins in cells. In specific embodiments, the disclosurerelates to compounds disclosed herein, such as2,4-diamino-1,3,5-triazine derivatives or salts thereof, andcompositions including such compounds, as well as their methods of use.The 2,4-diamino-1,3,5-triazine derivatives can be, for example,2,4-diamino-6-vinyl-1,3,5-triazine (Compound 12) or compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine.In certain embodiments, the composition further comprises a bonemorphogenetic protein and/or another growth factor. Typically, the bonemorphogenetic protein is BMP-2 or BMP-7. In certain embodiments, thegraft composition comprises calcium phosphates and/or bone granules,hydroxyapatite and/or beta-tricalcium phosphate, alpha-tricalciumphosphate, polysaccharides or combinations thereof. Crushed bonegranules, typically obtained from the subject, are optionally added tothe graft composition.

In some embodiments, the 2,4-diamino-1,3,5-triazine derivatives haveformula I:

or a salt thereof, wherein

if

is a single bond then R³ is carbocyclyl, aryl, or heterocyclyl whereinR³ is optionally substituted with one or more the same or different R⁴,or R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, or heterocyclylwherein R³ is optionally substituted with one or more the same ordifferent R⁴, R² is hydrogen or alkyl, such as methyl, and R¹ ishydrogen or alkyl; or

if

is a double bond then R¹ is alkyl, alkenyl, carbocyclyl, aryl, orheterocyclyl wherein R¹ is optionally substituted with one or more thesame or different R⁴; and R² and R³ are absent;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methyl sulfinyl, ethyl sulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments,

is a single bond, R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, orheterocyclyl wherein R³ is optionally substituted with one or more thesame or different R⁴, and R¹ and R² are each the same or differentalkyl, such as methyl. In certain embodiments, R⁴ is halogen, alkyl,alkoxy, or hydroxy.

In some embodiments, the disclosure relates to a bone graft compositionscomprising a compound disclosed herein, such as a2,4-diamino-1,3,5-triazine derivative, or a salt thereof, and a graftmatrix. Typically, the matrix comprises a collagen sponge and/or acompression resistant type I collagen and calcium phosphates. In otherembodiments, the matrix is a hydrogel.

In some embodiments, the disclosure relates to kits comprising a graftcomposition, a compound disclosed herein, such as a2,4-diamino-1,3,5-triazine derivative, or a salt thereof, and a graftmatrix. In certain embodiments, the kits further comprise a bonemorphogenetic protein and/or another growth factor. In certainembodiments, the kits further comprise a transfer device, such as asyringe or pipette.

In some embodiments, the disclosure relates to methods of generatingBMP-mediated osteoblasts comprising administering an effective amount ofcompound(s) disclosed herein to cells capable of osteoblasticdifferentiation, such as mesenchymal stem cells and pre-osteoblasticcells.

In some embodiments, the disclosure relates to methods of forming boneor cartilage, comprising implanting a graft composition comprising acompound disclosed herein, such as a 2,4-diamino-1,3,5-triazinederivative, or a salt thereof, in a subject under conditions such thatbone or cartilage forms in the graft. Typically, the subject has a voidin the bony structure wherein the graft composition is implanted in thevoid. In certain embodiments, the void is in a bone selected from anextremity, maxilla, mandible, pelvis, spine and/or cranium. In certainembodiments, the void is a result of surgical removal of bone. Incertain embodiments, the void is between bone and an implanted medicaldevice. In another embodiment, the method further comprises the step ofsecuring movement of bone structure with a fixation system, and removingthe system after bone forms in the implanted graft.

In certain embodiments, the disclosure relates to uses of2,4-diamino-1,3,5-triazine derivatives or salts thereof for cartilageregeneration e.g., between intervertebral disc and articular, jaw,elbow, knee, ankle, wrist, and hip joints. Methods contemplate oraladministration, intravenous administration, or direct injection at thedesired site(s) of the subject.

In some embodiments, the disclosure relates to methods of performingspinal fusion comprising implanting a bone graft composition comprisinga compound disclosed herein, such as a 2,4-diamino-1,3,5-triazinederivative, or a salt thereof, configured to grow bone between twovertebrae of a subject. In certain embodiments, the composition furthercomprises a bone morphogenetic protein and/or another growth factor. Ina typical embodiment, the subject is diagnosed with degenerative discdisease or has symptoms of back pain.

In some embodiments, the disclosure relates to methods of inserting aprosthetic device or anchor, comprising exposing a bone; and implantinga graft composition comprising a compound disclosed herein, such as a2,4-diamino-1,3,5-triazine derivative, or a salt thereof, in contactwith the bone. In certain embodiments, one implants the prostheticdevice or anchor in the graft composition. In certain embodiments, thecomposition further comprises a bone morphogenetic protein and/oranother growth factor.

In some embodiments, the disclosure relates to pharmaceuticalcompositions comprising compounds disclosed herein, such as2,4-diamino-1,3,5-triazine derivatives, or a pharmaceutically acceptablesalts thereof. In certain embodiments, the composition further comprisesa bone morphogenetic protein and/or another growth factor. In certainembodiments, the pharmaceutical composition is formulated to releaseover a 12 hour, 1 day, 3 day, 5 day, 7 day, two week, or one monthperiod.

In certain embodiments, the disclosure relates to methods of preventingor treating a bone fracture, comprising administering a pharmaceuticalcomposition comprising a compound disclosed herein, such as a2,4-diamino-1,3,5-triazine derivative, or a pharmaceutically acceptablesalt thereof, to a subject at risk for, exhibiting symptoms of, ordiagnosed with a bone fracture. In certain embodiments, the compositionfurther comprises a bone morphogenetic protein and/or another growthfactor. In certain embodiments, the administration is localized. Incertain embodiments, administration is achieved through oral delivery,intravenous delivery, parenteral delivery, intradermal delivery,percutaneous delivery, or subcutaneous delivery. In some embodiments,the method further comprises the step of exposing the bone fracture topulsed electromagnetic fields. In further embodiments, the subject isdiagnosed with a long bone shaft fracture such as a tibia or femurfracture corrected with intramedullary nail fixation.

In some embodiments, the disclosure relates to methods of preventing ortreating a bone degenerative disease, comprising administering apharmaceutical composition comprising a compound disclosed herein, suchas a 2,4-diamino-1,3,5-triazine derivative, or a pharmaceuticallyacceptable salt thereof, to a subject at risk for, exhibiting symptomsof, or diagnosed with a bone degenerative disease. In certainembodiments, the composition further comprises a bone morphogeneticprotein and/or another growth factor. In certain embodiments, theadministration is systemic, or administration is achieved through oraldelivery, intravenous delivery, parenteral delivery, intradermaldelivery, percutaneous delivery, or subcutaneous delivery. In someembodiments, the disease is osteoporosis, osteitis deformans, bonemetastasis, multiple myeloma, primary hyperparathyroidism, orosteogenesis imperfecta.

In some embodiments, the disclosure relates to methods for decreasingthe time required to form new bone in the presence of a bonemorphogenetic protein, comprising co-administering at least one compounddisclosed herein, such as a 2,4-diamino-1,3,5-triazine derivative, or asalt thereof.

In some embodiments, the disclosure relates to a process for engineeringbone tissue comprising combining a compound disclosed herein, such as a2,4-diamino-1,3,5-triazine derivative, or a salt thereof, and optionallya bone morphogenetic protein, with a cell selected from the groupconsisting of osteogenic cells, pluripotent stem cells, mesenchymalcells, and embryonic stem cells.

In some embodiments, the disclosure relates to compositions and methodsdisclosed herein wherein the 2,4-diamino-1,3,5-triazine derivative is a1-(phenyl)-5-isopropyl-biguanide or a salt thereof optionallysubstituted with one or more substituents. In certain embodiments, the2,4-diamino-1,3,5-triazine derivative has formula II:

or a salt thereof wherein

R¹ is hydrogen or alkyl; R² is hydrogen or alkyl, such as methyl, and R³is alkyl, phenyl, benzyl, carbocyclyl, aryl, or heterocyclyl optionallysubstituted with one or more the same or different R⁴;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R³ is phenyl substituted with one or morehalogens.

In certain embodiments, the 1-(phenyl)-5-isopropyl-biguanide derivativeis proguanil, 1-(4-chlorophenyl)-5-isopropyl-biguanide hydrochloride orsalt thereof. These compounds can be used in promotion of bone growthand for the treatment of osteoporosis and other bone growth relateddiseases and disorders. In some embodiments, the disclosure relates tocompositions and methods disclosed herein wherein the2,4-diamino-1,3,5-triazine derivative is an aromatic triazine or anaromatic 1,3,5-triazine such as: 2,4-diamino-6-phenyl-1,3,5-triazine,

2-chloro-4,6-diamino-1,3,5-triazine,

6-methyl-1,3, 5-triazine-2,4-diamine,

2,4-diamino-1,3,5-triazine,

2,4-diamino-6-(2,3-xylyl)-1,3,5-triazine,

2,4-diamino-6-(m-tolyl)-1,3,5-triazine,

2,4-diamino-6-isobutyryl-1,3,5-triazine,

2,4-diamino-6-phenylacetyl-1,3,5-triazine,

4,6-diamino-2-hydroxy-1,3,5-triazine,

2-chloro-4,6-bis(ethylamino)-1,3,5-triazine,

4,6-dimethyl-1,3,5-triazin-2-amine,

6-methyl-1,3, 5-triazine-2,4-diamine,

2,4-diamino-6-(2-fluorophenyl)-1,3,5-triazine,

2,4,diamino-6-(3,5-difluorophenyl)-1,3,5-triazine,

2,4-diamino-6-(3-fluorophenyl)-1,3,5-triazine,

2,4-diamino-6-(4-bromophenyl)-1,3,5-triazine,

6-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine,

2,4-diamino-6-(4-methoxphenyl)-1,3,5-triazine,

2,4-diamino-6-(4-methylphenyl)-1,3,5-triazine,

2,4-diamino-6-(3-nitrophenyl)-1,3,5-triazine,

4,6-diamino-gamma-oxo-1,3,5-triazine-2-butyric acid,

4,6-diamino-gamma-oxo-1,3,5-triazine-2-butyronitrile,

2,4-diamino-6-vinyl-1,3,5-triazine,

6-propyl-1,3, 5-triazine-2,4-diamine,

6-isobutyl-1,3,5-triazine-2,4-diamine, or salts thereof.

In some embodiments, the disclosure relates to compositions and methodsdisclosed herein wherein the 2,4-diamino-1,3,5-triazine derivative is anon-aromatic triazine or a non-aromatic 1,3,5-triazine such as:

1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,1-isopropyl-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-isobutyl-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

6,6-dimethyl-1-(2-methylbutyl)-1,6-dihydro-1,3,5-triazine-2,4-diamine,

1-(cyclopropylmethyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2-methoxyethyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-benzyl-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-methoxybenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-methoxybenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-bromo-2-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2-bromo-4-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

6,6-dimethyl-1-(4-methyl-3-nitrophenyl)-1,6-dihydro-1,3,5-triazine-2,4-diamine,

1-(2,4-difluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2-fluoro-4-iodophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-bromo-2-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-chloro-4-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-chloro-2,5-dimethoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-chloro-2-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-chloro-2-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

5-chloro-2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)benzoicacid,

5-chloro-2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)-3-methylbenzonitrile,

1-(4-chloro-2-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-chloro-4-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(5-chloro-2,4-dimethoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-chloro-4-ethylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-chloro-5-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

2-chloro-4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)phenol,

1-(5-chloro-2-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

3-chloro-4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)-5-methylbenzonitrile,

1-(3-chloro-5-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2-chloro-5-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2-chloro-4-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(5-chloro-2-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(5-chloro-2-ethylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2-chloro-4-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

methyl3-chloro-4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)benzoate,

3-chloro-4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)phenol,

1-(2-chloro-6-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-fluoro-2-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

3-chloro-4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)benzonitrile,

1-(2-chloro-6-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-chloro-4-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

(5-chloro-2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)phenyl)methanol,

1-(4-chloro-3-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-chloro-4-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-bromo-2-fluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2,6-difluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3-bromo-4-fluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

4-((4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)methyl)-2-methoxyphenol,

1-(3-chloro-4-fluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(2,4-dimethoxybenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(5-bromo-2-fluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3,5-difluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(3,4-dimethoxybenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

6,6-dimethyl-1-(3-(trifluoromethyl)benzyl)-1,6-dihydro-1,3,5-triazine-2,4-diamine,

1-(3-fluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(4-fluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,

1-(5-bromo-2-fluorobenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,and

6,6-dimethyl-1-(3-nitrobenzyl)-1,6-dihydro-1,3,5-triazine-2,4-diamine orsalts thereof.

In certain embodiments, the 2,4-diamino-1,3,5-triazine derivative is acompound selected from the group consisting of:

1-benzyl-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(4-methoxybenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(3-methoxybenzyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(3-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(3-chloro-4-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(3-chloro-4-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(4-bromo-2-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(2-bromo-4-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

6,6-dimethyl-1-(4-methyl-3-nitrophenyl)-1,6-dihydro-1,3,5-triazine-2,4-diamine;

1-(3-chloro-4-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

1-(3-chloro-5-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;

2-chloro-4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yl)phenol;

1-(2-chloro-5-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;and

1-(2-chloro-4-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazineor salts thereof.

In certain embodiments, the disclosure relates to any of the2,4-diamino-1,3,5-triazine derivatives disclosed herein, substitutedwith one or more substituents.

In certain embodiments, the disclosure relates to methods of makingcompounds disclosed herein by mixing an amine and guanidine, guanidinesalt, or imine formed from a ketone or aldehyde to guanidine underconditions such that compounds disclosed herein are formed.

In certain embodiments, the disclosure relates to using2,4-diamino-1,3,5-triazine derivatives disclosed herein in theproduction of a medicament for the treatment or prevention of a bonedisease or other applications disclosed herein.

In certain embodiments, the disclosure relates to a bone graftcomposition comprising a bone growth-inducing amount of a2,4-diamino-1,3,5-triazine derivative or salt thereof, and apharmaceutically acceptable carrier.

In certain embodiments, the 2,4-diamino-1,3,5-triazine derivative hasformula I or salts thereof, wherein

(A) if

is a single bond, then R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl,or heterocyclyl optionally substituted with one or more the same ordifferent R⁴, R² is hydrogen or alkyl, and R¹ is hydrogen or alkyl; or

(B) if

is a double bond, then R¹ is alkyl, alkenyl, carbocyclyl, aryl, orheterocyclyl optionally substituted with one or more the same ordifferent R⁴, and R² and R³ are absent;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵ groups; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments,

is a single bond; R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, orheterocyclyl, optionally substituted with one or more of the same ordifferent R⁴groups; R² is hydrogen or alkyl; and R¹ is hydrogen oralkyl.

In certain embodiments, the 2,4-diamino-1,3,5-triazine derivative is a1-(phenyl)-5-isopropyl-biguanide or salt thereof optionally substitutedwith one or more substituents.

In certain embodiments, the bone graft composition further comprises agrowth factor.

In certain embodiments, the growth factor comprises a bone morphogeneticprotein.

In certain embodiments, the bone morphogenetic protein is selected fromthe group consisting BMP-2, BMP-7, BMP-6, BMP-9, and combinationsthereof.

In certain embodiments, the bone graft composition further comprises oneor more calcium phosphates.

In certain embodiments, the calcium phosphates are selected from thegroup consisting of hydroxyapatite, tricalcium phosphate, andcombinations thereof.

In certain embodiments, the pharmaceutically acceptable carriercomprises a matrix selected from the group consisting of collagens,hydrogels, and combinations thereof.

In certain embodiments, the disclosure relates to a kit comprising abone morphogenetic protein and the bone graft composition disclosedherein.

In certain embodiments, the disclosure relates to a method of formingbone or cartilage comprising implanting the bone graft compositiondisclosure herein in a subject under conditions such that bone orcartilage forms.

In certain embodiments, the bone graft composition further comprises agrowth factor.

In certain embodiments, the subject has a void in its bony structure andthe method comprises implanting the bone graft composition in the void.

In certain embodiments, the bony structure is selected from the groupconsisting of an extremity, maxilla, a mandible, a pelvis, a spine, acranium, or a combination thereof.

In certain embodiments, the method comprises implanting the bone graftcomposition between two vertebrae of the subject to grow bone betweenthe vertebrae.

In certain embodiments, the disclosure relates to a method of preventingor treating a bone fracture comprising administering a pharmaceuticalcomposition comprising a 2,4-diamino-1,3,5-triazine derivative or saltthereof to a subject at risk for, exhibiting symptoms of, or diagnosedwith a bone fracture.

In certain embodiments, the pharmaceutical composition further comprisesa growth factor.

In certain embodiments, the disclosure relates to a method of preventingor treating a bone degenerative disease comprising administering apharmaceutical composition comprising a 2,4-diamino-1,3,5-triazinederivative or pharmaceutically acceptable salt thereof to a subject atrisk for, exhibiting symptoms of, or diagnosed with a bone degenerativedisease.

In certain embodiments, the bone degenerative disease is selected fromthe group consisting of osteoporosis, osteitis deformans, bonemetastasis, multiple myeloma, primary hyperparathyroidism, andosteogenesis imperfecta.

In certain embodiments, the pharmaceutical composition further comprisesa growth factor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows data on ectopic bone induction by compound 12:2,4-diamino-6-vinyl-1,3,5-triazine and BMP-2.

FIG. 2 shows data on ectopic bone induction by compound 12:2,4-diamino-6-vinyl-1,3,5-triazine without BMP-2.

FIG. 3 illustrates important amino acid residues in WW2 domain ofSmurf1. Residues labeled in white boxes are important for structuralintegrity of beta sheets in the WW-domain. Residues labeled in pinkboxes are involved in natural target protein interaction commonly inWW-domains. Residues labeled in green boxes are unique to Smurf1interaction and confer binding specificity to its natural targets.

FIG. 4 shows the energetically favored 2D interaction map ofcompound-12-WW2-Molecule. CDOCKING was performed using compound-12 and6-key residues in Smurf1-WW2 domain (Y-V-H—R-T-F) as receptor bindingsites. In a ligand pose the residues, Asp21 and Arg25, participate inH-bonding.

FIG. 5A shows data on of the efficacy of various compounds to enhanceBMP-induced luciferase activity. Relative activities of a representativeset of candidate compounds that were selected by virtual screenings weretested in the luciferase reporter assay are shown. Compounds were testedat a concentration of 1.0 ug/ml while BMP-2 was used at 1.0 ng/ml.

FIG. 5B shows data on of the efficacy of various compounds to enhanceBMP-induced luciferase activity. Relative activities of a representativeset of candidate compounds that were selected by virtual screenings weretested in the luciferase reporter assay are shown. Compounds were testedat a concentration of 1.0 ug/ml while BMP-2 was used at 1.0 ng/ml.

FIG. 6 shows data of potentiation of BMP-2-induced ALP mRNA levels withcompound 12.

FIG. 7 shows data on potentiation of BMP-2-induced osteocalcin geneexpression with compound 12.

FIG. 8 shows data on potentiation of BMP-induced alkaline phosphataseactivity with compound 12.

FIG. 9 shows data on the activity of 2,4-diamino-1,3,5-triazinederivatives in promoting

BMP-induced of alkaline phosphatase activity. Compound A is2,4-diamino-6-phenyl-1,3,5-triazine, B is2-Chloro-4,6-diamino-1,3,5-triazine, C is6-methyl-1,3,5-triazine-2,4-diamine, D is 2,4-diamino-1,3,5-triazine, Eis 2,4-diamino-6-(2,3-xylyl)-1,3,5-triazine, F is2,4-diamino-6-(m-tolyl)-1,3,5-triazine, G is2,4-diamino-6-isobutyryl-1,3,5-triazine, H is2,4-diamino-6-phenylacetyl-1,3,5-triazine, I is4,6-diamino-2-hydroxy-1,3,5-triazine, J is2-chloro-4,6-bis(ethylamino)-1,3,5-triazine, K is4,6-dimethyl-1,3,5-triazin-2-amine, L is6-methyl-1,3,5-triazine-2,4-diamine, M is2,4-Diamino-6-(2-fluorophenyl)-1,3,5-triazine, N is2,4,Diamino-6-(3,5-difluorophenyl)-1,3,5-triazine, P is2,4-diamino-6-(3-fluorophenyl)-1,3,5-triazine, Q is2,4-diamino-6-(4-bromophenyl)-1,3,5-triazine, R is6-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine, S is2,4-diamino-6-(4-methoxphenyl)-1,3,5-triazine, T is2,4-diamino-6-(4-methylphenyl)-1,3,5-triazine, U is2,4-diamino-6-(3-nitrophenyl)-1,3,5-triazine, V is4,6-diamino-gamma-oxo-1,3,5-triazine-2-butyric acid, W is4,6-diamino-gamma-oxo-1,3,5-triazine-2-butyronitrile, X (or compound12X) is1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,and compound 12 is 2,4-diamino-6-vinyl-1,3,5-triazine.

FIG. 10 shows dose response for compound 12X as determined bypotentiation of BMP-induced alkaline phosphatase activity.

FIG. 11 shows ADMET properties from 12 to 12X as determined by DiscoveryStudio 2.5.5.version (Accelrys, Inc, San Diego, Calif.).

FIG. 12 shows the molecular design of compounds that exhibit osteogenicactivity without tendency for toxicity.

FIG. 13A shows data for compound 12 on bone inducing activity in afracture model using a gap size, bone in gap, and mineralized callusscoring systems.

FIG. 13B shows data for compound 12 on bone inducing activity in afracture model using a gap size, bone in gap, and mineralized callusscoring systems.

FIG. 13C shows data for compound 12 on bone inducing activity in afracture model using a gap size, bone in gap, and mineralized callusscoring systems.

FIG. 13D shows data for compound 12 on bone inducing activity in afracture model using a gap size, bone in gap, and mineralized callusscoring systems.

FIG. 13E shows data for compound 12 on bone inducing activity in afracture model using a gap size, bone in gap, and mineralized callusscoring systems.

FIG. 14A shows data of the fold increase in BMP-induced ALP activity forcompounds disclosed herein. Compound 12 is2,4-diamino-6-vinyl-1,3,5-triazine; 12X is1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,12X-33 is1-(3-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;12X-35 is1-(3-chloro-4-methoxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;12X-81 is1-(3-chloro-4-methylphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;12X-83 is1-(3-chloro-5-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;12X-86 is1-(3-chloro-4-hydroxyphenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine;and 12X-329 is1-(3-chloro-4-fluorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine.

FIG. 14B shows data of the fold increase in BMP-induced ALP activity forcompounds disclosed herein.

FIG. 15 shows data on the induction of mineralization in ROB cells.

FIG. 16 shows a gel related to homologous binding competition withligand blotting of purified Smurf1 with unlabeled 12X-86 and12X-86-I¹²⁵.

FIG. 17A illustrates the schematic synthetic preparation routes ofcertain embodiments. Substitution of appropriate starting materials maybe used to prepare derivatives that are not commercially available.

FIG. 17B illustrates the schematic synthetic preparation routes ofcertain embodiments. Substitution of appropriate starting materials maybe used to prepare derivatives that are not commercially available.

FIG. 17C illustrates the schematic synthetic preparation routes ofcertain embodiments. Substitution of appropriate starting materials maybe used to prepare derivatives that are not commercially available.

FIG. 17D illustrates the schematic synthetic preparation routes ofcertain embodiments. Substitution of appropriate starting materials maybe used to prepare derivatives that are not commercially available.

DETAILED DISCUSSION Terms

“Ossification” refers to the process of laying down new bone by cellscalled osteoblasts. The term includes the growth in healing bonefractures treated by cast or by open reduction and stabilization bymetal plate and screws. Ossification can also result in the formation ofbone tissue in an extraskeletal location.

The term “bone morphogenetic protein” or “BMP” refers to any one of thefamily of growth factors or fragments thereof with the ability to inducethe formation of bone and/or cartilage. The BMP receptors are typicallyserine-threonine kinases. It is not intended that BMP refer to anyparticular protein sequence and may or may not have certainglycosylation patterns attached thereto provided that the molecule hassufficient structural homology to any one of the known BMPs describedbelow and retains some functional ability to promote bone growth,cartilage growth, or osteoblast differentiation. BMPs may be isolatedfrom natural or non-natural sources, such as, but not limited to,recombinant or synthetic methods. References to BMPs generally or aspecific BMP, e.g, BMP-2, includes recombinant or synthetically isolatedversions unless otherwise provide for herein. Combinations of BMPs arecontemplated. BMP-2 is known to induce bone and cartilage formation andplay a role in osteoblast differentiation. BMP-3 is known to induce boneformation. BMP-4 is known to regulate the formation of teeth, limbs andbone from mesoderm and play a role in fracture repair. BMP-5 is known tofunction in cartilage development. BMP-6 is known to play a role injoint integrity and bone formation/repair. BMP-7 and BMP-9 are known toplay a role in osteoblast differentiation. BMP-1 is a knownmetalloprotease that acts on procollagen I, II, and III and is involvedin cartilage development.

As used herein, the term “derivative” refers to a structurally similarcompound that retains sufficient functional attributes of the identifiedanalogue. A derivative of 2,4-diamino-1,3,5-triazine derivatives may becompounds, such as 2,4-diamino-6-vinyl-1,3,5-triazine or compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine.The derivative may be structurally similar because it is lacking one ormore atoms, substituted, a salt, in different hydration/oxidationstates, or because one or more atoms within the molecule are switched,such as, but not limited to, replacing a oxygen atom with a sulfur atomor replacing a amino group with a hydroxy group. The derivative may be aprodrug. Derivatives can be prepare by any variety of synthetic methodsor appropriate adaptations presented in synthetic or organic chemistrytext books, such as those provide in March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) MichaelB. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F.Tietze.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents.” The molecule may be multiplysubstituted. In the case of an oxo substituent (“═O”), two hydrogenatoms are replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl,carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb,—NRaC(═O)NRaNRb, —NRaC(═O)ORb,—NRaSO2Rb,—C(═O)Ra,—C(═O)ORa,—C(═O)NRaRb,—OC(═O)NRaRb,—ORa,—SRa,—SORa,—S(═O)2Ra,—OS(═O)2Ra and —S(═O)2ORa. Ra and Rb in thiscontext may be the same or different and independently hydrogen, halogenhydroxy, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino,carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl,aryl, arylalkyl, heteroaryl, heteroarylalkyl.

The term “optionally substituted,” as used herein, means thatsubstitution is optional and therefore it is possible for the designatedatom to be unsubstituted.

As used herein, “subject” refers to any animal, preferably a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “calcium phosphate(s)” refers to mineralscontaining calcium ions together with orthophosphates, metaphosphates orpyrophosphates and optionally hydroxide ions. Tricalcium phosphate is acalcium phosphate with formula Ca₃(PO₄)₂. The common mineral apatite hasthe basic formula Ca₅(PO₄)₃X, where X is a ion, typically a halogen orhydroxide ion, or a mixture. Hydroxyapatite refers to apatite where X ismainly hydroxide ion.

When used in reference to compound(s) disclosed herein, “salts” refer toderivatives of the disclosed compound(s) where the parent compound ismodified making acid or base salts thereof. Examples of salts include,but are not limited to, mineral or organic acid salts of basic residuessuch as amines, alkylamines, or dialkylamines; alkali or organic saltsof acidic residues such as carboxylic acids; and the like.

As used herein, “alkyl” means a noncyclic straight chain or branched,unsaturated or saturated hydrocarbon such as those containing from 1 to10 carbon atoms. Representative saturated straight chain alkyls includemethyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl,n-nonyl, and the like; while saturated branched alkyls includeisopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Unsaturated alkyls contain at least one double or triple bond betweenadjacent carbon atoms (referred to as an “alkenyl” or “alkynyl”,respectively). Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like; while representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as“carbocycles” or “carbocyclyl” groups. Representative saturatedcarbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and the like; while unsaturated carbocycles include cyclopentenyl andcyclohexenyl, and the like.

“Heterocarbocycles” or heterocarbocyclyl” groups are carbocycles whichcontain from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur which may be saturated or unsaturated (but notaromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfurheteroatoms may be optionally oxidized, and the nitrogen heteroatom maybe optionally quaternized. Heterocarbocycles include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

“Aryl” means an aromatic carbocyclic monocyclic or polycyclic ring suchas phenyl or naphthyl. Polycyclic ring systems may, but are not requiredto, contain one or more non-aromatic rings, as long as one of the ringsis aromatic.

As used herein, “heteroaryl” or “heteroaromatic” refers an aromaticheterocarbocycle having 1 to 4 heteroatoms selected from nitrogen,oxygen and sulfur, and containing at least 1 carbon atom, including bothmono- and polycyclic ring systems. Polycyclic ring systems may, but arenot required to, contain one or more non-aromatic rings, as long as oneof the rings is aromatic. Representative heteroaryls are furyl,benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl,isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It iscontemplated that the use of the term “heteroaryl” includes N-alkylatedderivatives such as a 1-methylimidazol-5-yl substituent.

As used herein, “heterocycle” or “heterocyclyl” refers to mono- andpolycyclic ring systems having 1 to 4 heteroatoms selected fromnitrogen, oxygen and sulfur, and containing at least 1 carbon atom. Themono- and polycyclic ring systems may be aromatic, non-aromatic ormixtures of aromatic and non-aromatic rings. Heterocycle includesheterocarbocycles, heteroaryls, and the like.

“Alkylthio” refers to an alkyl group as defined above attached through asulfur bridge. An example of an alkylthio is methylthio, (i.e.,—S—CH3).

“Alkoxy” refers to an alkyl group as defined above attached through anoxygen bridge. Examples of alkoxy include, but are not limited to,methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy,n-pentoxy, and s-pentoxy. Preferred alkoxy groups are methoxy, ethoxy,n-propoxy, propoxy, n-butoxy, s-butoxy, t-butoxy.

“Alkylamino” refers an alkyl group as defined above attached through anamino bridge. An example of an alkylamino is methylamino, (i.e.,—NH—CH₃).

“Alkanoyl” refers to an alkyl as defined above attached through acarbonyl bride (i.e., —(C═O)alkyl).

“Alkylsulfonyl” refers to an alkyl as defined above attached through asulfonyl bridge (i.e., —S(═O)₂alkyl) such as mesyl and the like, and“Arylsulfonyl” refers to an aryl attached through a sulfonyl bridge(i.e.,—S(═O)₂aryl).

“Alkylsulfamoyl” refers to an alkyl as defined above attached through asulfamoyl bridge (i.e.,—S(═O)₂NHalkyl), and an “Arylsulfamoyl” refers toan alkyl attached through a sulfamoyl bridge (i.e.,—S(═O)₂NHaryl).

“Alkylsulfinyl” refers to an alkyl as defined above with the indicatednumber of carbon atoms attached through a sulfinyl bridge (i.e.—S(═O)alkyl).

The terms “halogen” and “halo” refer to fluorine, chlorine, bromine, andiodine.

The term “bone graft composition” refers to materials that aresubstantially physiologically compatible when residing in bone area,void, or exterior site. In certain embodiments, the bone graftcomposition may be a bone graft matrix such as a collagen sponge or amixture of polymers and salts.

Compounds

In certain embodiments, the disclosure relates to2,4-diamino-1,3,5-triazine derivatives having formula I:

or a salt thereof, wherein

if

is a single bond then R³ is carbocyclyl, aryl, or heterocyclyl whereinR³ is optionally substituted with one or more the same or different R⁴,or R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, or heterocyclylwherein R³ is optionally substituted with one or more the same ordifferent R⁴, R² is hydrogen or alkyl, such as methyl, and R¹ ishydrogen or alkyl; or

if

is a double bond then R¹ is alkyl, alkenyl, carbocyclyl, aryl, orheterocyclyl wherein R¹ is optionally substituted with one or more thesame or different R⁴; and R² and R³ are absent;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments,

is a single bond, R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, orheterocyclyl wherein R³ is optionally substituted with one or more thesame or different R⁴, and R¹ and R² are each the same or differentalkyl, such as methyl.

In certain embodiments, R¹ and R² are alkyl.

In certain embodiments, R⁴ is halogen, alkyl, alkoxy, or hydroxy.

In certain embodiments, R⁴ is chloro.

In certain embodiments, R³ is phenyl or benzyl wherein R³ is substitutedwith one or more halogens, alkyl, hydroxy or alkoxy.

In certain embodiments, R³ is 3-chlorophenyl optionally substituted withone or more halogens, alkyl, hydroxy or alkoxy.

In certain embodiments, the 2,4-diamino-1,3,5-triazine derivative hasformula I or salts thereof, wherein

(A) if

is a single bond, then R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl,or heterocyclyl optionally substituted with one or more the same ordifferent R⁴, R² is hydrogen or alkyl, and R¹ is hydrogen or alkyl; or

(B) if

is a double bond, then R¹ is alkyl, alkenyl, carbocyclyl, aryl, orheterocyclyl optionally substituted with one or more the same ordifferent R⁴, and R² and R³ are absent;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵ groups; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methyl sulfamoyl, N-ethyl sulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In some embodiments, the 2,4-diamino-1,3,5-triazine derivatives haveformula I or salts thereof, wherein

if

is a single bond then R³ is carbocyclyl, aryl, or heterocyclyl whereinR³ is optionally substituted with one or more the same or different R⁴;

R² is hydrogen or alkyl, such as methyl, and R¹ is hydrogen or alkyl; or

if

is a double bond then R¹ is alkyl, alkenyl, carbocyclyl, aryl, orheterocyclyl wherein R¹ is optionally substituted with one or more thesame or different R⁴; and R² and R³ are absent;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methyl sulfinyl, ethyl sulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, the disclosure relates to2,4-diamino-1,3,5-triazine derivatives having formula IA:

or a salt thereof, wherein

R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, or heterocyclyl whereinR³ is optionally substituted with one or more the same or different R⁴;

R² is hydrogen or alkyl;

R¹ is hydrogen or alkyl;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methyl sulfinyl, ethyl sulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl, carbocyclyl,aryl, or heterocyclyl.

In certain embodiments, R⁴ is halogen, alkyl, alkoxy, or hydroxy.

In certain embodiments, R⁴ is chloro.

In certain embodiments, R³ is phenyl or benzyl wherein R³ is substitutedwith one or more halogens, alkyl, hydroxy or alkoxy.

In certain embodiments, R³ is 3-chlorophenyl optionally substituted withone or more halogens, alkyl, hydroxy or alkoxy.

In certain embodiments, R¹ and R² are alkyl.

In certain embodiments, the disclosure relates to2,4-diamino-1,3,5-triazine derivatives having formula IB:

or a salt thereof, wherein

R¹ is alkyl, alkenyl, carbocyclyl, aryl, or heterocyclyl optionallysubstituted with one or more the same or different R⁴;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkylsulfonyl, arylsulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methyl sulfinyl, ethyl sulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R⁴ is halogen, alkyl, alkoxy, or hydroxy.

In certain embodiments, R¹ is phenyl or benzyl wherein R¹ is substitutedwith one or more halogens, alkyl, hydroxy or alkoxy.

In certain embodiments, the 2,4-diamino-1,3,5-triazine derivatives haveformula II:

or a salt thereof wherein

R¹ is hydrogen or alkyl; R² is hydrogen or alkyl, such as methyl, and R³is alkyl, phenyl, benzyl, carbocyclyl, aryl, or heterocyclyl wherein R³is optionally substituted with one or more the same or different R⁴;

R⁴ is alkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl,carboxy, carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino,alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, orheterocyclyl, wherein R⁴ is optionally substituted with one or more, thesame or different, R⁵; and

R⁵ is halogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methylsulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethylsulfamoyl,N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethylsulfamoyl,carbocyclyl, aryl, or heterocyclyl.

In certain embodiments, R⁴ is halogen, alkyl, alkoxy, or hydroxy.

In certain embodiments, R⁴ is chloro.

In certain embodiments, R³ is phenyl or benzyl wherein R³ is substitutedwith one or more halogens, alkyl, hydroxy or alkoxy.

In certain embodiments, R³ is 3-chlorophenyl optionally substituted withone or more halogens, alkyl, hydroxy or alkoxy.

In certain embodiments, R³ is phenyl or benzyl wherein R³ is substitutedwith one or more halogens, alkyl, hydroxy or alkoxy.

In certain embodiments, R¹ and R² are alkyl.

Evaluations of Compound Activity

Potentiating effect of compound 12; (2,4-diamino-6-vinyl-1,3,5-triazine)in BMP-induced in vivo bone-formation in rat model was evaluated (seeFIG. 1). Compound 12 is capable of inducing bone-formation without anyBMP-2 in rat model (see FIG. 2). The compound is selected by virtualscreening of chemical databases using the Ludi docking and scoringfunctions. FIG. 3 shows the key amino acid residues of Smurf1-WW2 domaininvolved in determining the unique beta sheet (secondary structure) aswell as the residues required for common ligand interaction (residueslabeled pink, FIG. 3). Important amino acid residues for Smurf1-specificinteraction to the natural target proteins were also identified(residues labeled in green box, FIG. 3). The interaction map forcompound binding to WW2 domain of Smurf1 is shown in FIG. 4.

Derivatives with desired attributes can be evaluated using the proceduredescribed below. It has been identified that2,4-diamino-6-vinyl-1,3,5-triazine and compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazinepotentiate the effects of BMP-2 in inducing transdifferentiation ofC2Cl2 myoblasts into the osteoblastic phenotype. The enhancement ofBMP-2 activity by 2,4-diamino-6-vinyl-1,3,5-triazine was characterizedby evaluating a BMP-specific reporter activity and by monitoring theBMP-2-induced expression of mRNA for osteocalcin and alkalinephosphatase which are widely accepted marker genes of osteoblastdifferentiation. The compound enhanced BMP-2-induced activity ofalkaline phosphataseand exhibits a dose-dependent activity in inducingosteoblastic differentiation of myoblastic C2Cl2 cells even whenmultiple markers of the osteoblastic phenotype were monitored inparallel.

A cell-based method was optimized to monitor BMP-2 induced responses.The mouse-derived C2Cl2 myoblasts served as an experimentally tractablemodel system for investigating the molecular basis oftransdifferentiation towards the osteoblastic phenotype. One can monitortranscriptional activity driven by activated Smad1 in C2Cl2 cells in acell-based assay utilizing a Smad1-specific luciferase reporter plasmidcontaining a multimerized -GCCG- motif (9 copies). Okada et al. (2009)Cell Biochem Funct. 27: 526-534. The BMP-specific Smad1-driven 9×GCCGreporter construct has been widely used to assay BMP activity in manycell types at a typical concentration of 50-100 ng/ml of BMP-2. Toselect a sub-optimal dose of BMP-2 for studying the potentiating effectof test compounds, the reporter assay was performed with lower BMP-2concentrations ranging from 0.25 to 25 ng/ml. A concentration range ofBMP-2 required for activating the reporter assay was established. Theresults from this experiment allowed selection of a sub-optimal dose(1.0 ug/ml) of BMP-2 to assess the potentiating effects to testcompounds in subsequent experiments.

The compound 2,4-diamino-6-vinyl-1,3,5-triazine potentiates BMP-2induced Smad1-driven luciferase reporter activity. Compounds were testedexperimentally for their ability to potentiate BMP-2 activity in ourluciferase reporter assay using C2Cl2 cells (FIGS. 5A and 5B). Thesolvent dimethylsulfoxide (DMSO) controls showed only basal activitysimilar to no treatment controls. The DMSO solvent concentration of0.01% (v/v) was not toxic to cells as determined by cell number, totalprotein amount, and cell phenotype consistent with the literature. Atconcentrations higher than 1.5 ug/ml, 2,4-diamino-6-vinyl-1,3,5-triazinecaused lifting of some cells from the plates, thus reducing the numberof cells at the end of the experiment. Compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazineis predicted to have a lower liver toxicity index based on “in silica”ADMET profiling (Discovery Studio 2.5.5, Accelrys, Inc., San Diego,Calif.) of the hepatotoxicity probability (Table 1).

TABLE 1 CDOCKER-Energy Hepatotoxicity Comp. Formal Name (negative value)Probability 12 6-vinyl-1,3,5-triazine-2,4-diamine 31.02 0.89 12X1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2- 19.19 0.31dimethyl-1,3,5-triazine 12X-1-(3-chloro-4-methylphenyl)-4,6-diamino-1,2- 20.52 0.304 81dihydro-2,2-dimethyl-1,3,5-triazine X101-(2,5-dimethylphenyl)-4,6-diamino-1,2-dihydro- 22.07 0.2252,2-dimethyl-1,3,5-triazine X131-(4-chloro-2-methylphenyl)-4,6-diamino-1,2- 20.48 0.278dihydro-2,2-dimethyl-1,3,5-triazine

Effectiveness of 2,4-diamino-6-vinyl-1,3,5-triazine on potentiatingBMP-2 activity was determined over the concentration range from 0.125 to1.0 ug/ml while keeping the BMP-2 concentration at 1 ng/ml in theluciferase reporter assay. It caused a dose-dependent enhancement of theluciferase activity with an optimum enhancement of 3.8-fold (P<0.05)observed at a concentration of 1.0 μg/ml when compared to BMP-2 alone.The EC₅₀ value of 2.6 uM was calculated from the Hill plot. The EC₅₀value was generated from fitted curves by solving for x-intercept at the50% activity level of the Y-intercept. After assay optimization, whichincluded determining optimal BMP-2 concentration, plating density ofcells, and compound incubation time, the promoter assay was shown todisplay the dynamic range and reproducibility required for a screeningassay.

BMP-2 induced alkaline phosphatase and osteocalcin mRNA levels areenhanced by 2,4-diamino-6-vinyl-1,3,5-triazine. Effectiveness of2,4-diamino-6-vinyl-1,3,5-triazine was determined over the concentrationrange from 0.125 to 1.0 ug/ml while keeping the BMP-2 concentrationconstant at 20 ng/ml by determining alkaline phosphatase mRNA levels(FIG. 6). It caused a dose-dependent increase in the BMP-inducedalkaline phosphatase mRNA level with the maximal 3.5-fold increase(P<0.05) compared to BMP-2 alone. BMP-2 alone induced a 43-fold or51-fold increase in alkaline phosphatase mRNA in the absence or thepresence of DMSO (0.01%), respectively, when compared to the ‘notreatment’ control.

Data for additional compounds that are derivatives of compound 12 isprovided in FIG. 9. Compound A is 2,4-diamino-6-phenyl-1,3,5-triazine, Bis 2-Chloro-4,6-diamino-1,3,5-triazine, C is6-methyl-1,3,5-triazine-2,4-diamine, D is 2,4-diamino-1,3,5-triazine, Eis 2,4-diamino-6-(2,3-xylyl)-1,3,5-triazine, F is2,4-diamino-6-(m-tolyl)-1,3,5-triazine, G is2,4-diamino-6-isobutyryl-1,3,5-triazine, H is2,4-diamino-6-phenylacetyl-1,3,5-triazine, I is4,6-diamino-2-hydroxy-1,3,5-triazine, J is2-chloro-4,6-bis(ethylamino)-1,3,5-triazine, K is4,6-dimethyl-1,3,5-triazin-2-amine, L is6-methyl-1,3,5-triazine-2,4-diamine, M is2,4-Diamino-6-(2-fluorophenyl)-1,3,5-triazine, N is2,4,Diamino-6-(3,5-difluorophenyl)-1,3,5-triazine, P is2,4-diamino-6-(3-fluorophenyl)-1,3,5-triazine, Q is2,4-diamino-6-(4-bromophenyl)-1,3,5-triazine, R is6-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine, S is2,4-diamino-6-(4-methoxyphenyl)-1,3,5-triazine, T is2,4-diamino-6-(4-methylphenyl)-1,3,5-triazine, U is2,4-diamino-6-(3-nitrophenyl)-1,3,5-triazine, V is4,6-diamino-gamma-oxo-1,3,5-triazine-2-butyric acid, W is4,6-diamino-gamma-oxo-1,3,5-triazine-2-butyronitrile, 12X is1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,and Compound 12 is 2,4-diamino-6-vinyl-1,3,5-triazine.

The effectiveness of 2,4-diamino-6-vinyl-1,3,5-triazine in increasingBMP-2-induced osteocalcin gene expression was determined over theconcentration range from 0.125 to 1.0 ug/ml while keeping the BMP-2concentration constant at 20 ng/ml. See FIG. 7. The compound caused adose-dependent increase in the BMP-induced osteocalcin mRNA level with amaximal 4.4-fold increase (P<0.05) compared to BMP-2 alone when observedat a compound concentration of 1.0 μg/ml. BMP-2 alone induced a 9-foldor 7-fold increase in osteocalcin mRNA in the absence or the presence ofDMSO (0.01%), respectively, when compared to ‘no treatment control’. Thecompound alone showed no significant effect in the absence of BMP-2. AnEC50 value in the range of 1.5 to 5.3 uM was estimated for theexpression of both alkaline phosphatase and osteocalcin mRNAs.

BMP-2 induced ALP enzyme activity is enhanced by2,4-diamino-6-vinyl-1,3,5-triazine. In evaluating enhancement of theBMP-induced alkaline phosphatase activity, concentrations of2,4-diamino-6-vinyl-1,3,5-triazine ranged of 0.125 to 1.0 μg/ml whilekeeping the BMP-2 concentration constant at 50 ng/ml. The ALP enzymeactivity assay demonstrated that the compound dose-dependently enhancedthe BMP-2 induced ALP activity (FIG. 8). The peak activity of themaximal 4.8-fold increase (P<0.001) was observed at a compoundconcentration of 1.0 μg/ml when compared to BMP-2 alone. An EC50 valueof 3.7 uM was estimated for the activity of alkaline phosphatase. Theseresults support that 2,4-diamino-6-vinyl-1,3,5-triazine enhancesBMP-induced osteoblastic responses in cells.

The compound 2,4-diamino-6-vinyl-1,3,5-triazine elevated theBMP-2-induced response significantly even at a dose of 0.1 μg/ml. Theeffective dose of compound remained consistent (0.275-1.0 ug/ml) amongmultiple assays. This suggested that the biological markers in C2Cl2cells that we chose to investigate are tightly controlled by the sameBMP-signaling pathway. All these studies suggest that the compound orderivatives may be useful in potentiating the BMP-2 responsiveness ofcells.

The in silico ADMET (Absorption, Distribution, Metabolism, Excretion andToxicity) profiling was performed on compounds using Discovery Studio2.5.5 (Accelrys, Inc., San Diego, Calif.). The compound 12 showedsatisfactory profiles for various ADMET properties except that it showeda higher value (0.89) for hepatotoxicity probability indicating that thecompound may be hepatotoxic. In order to find compounds with reduced orno hepatotoxic tendency, compounds were identified on the basis ofsimilarity search using ChemNavigator (Sigma-Aldrich) and Hit2Leadprograms. The binding affinity of various compounds was determined byperforming in silico CDOCKINGs. Both Compound 12 and Compound 12X showedaffinity in binding to same target residues (Asp and Arg) in WW2 domainof Smurf1 mediated by two H-bonds. After screening compounds furtherwith a cell-based alkaline phosphatase assay, compound 12X wasidentified as a more efficacious derivative (FIG. 9). Compound 12Xshowed a hepatotoxicity probability value of 0.31 (Table 1). Thebiological activity of compound 12X by was confirmed determining itsactivity at various doses as shown in FIG. 10.

Based on theoretical binding properties, biological activity and insilico ADMET profiles of Compound 12 and Compound 12X, additionalcomputational screenings were performed. These efforts yieldedadditional compounds (e.g., 12X2, 12X10, and 12X13). These compoundsshowed no hepatotoxic tendency. Certain of these compounds are shown inFIG. 12.

Growth Factors

In some embodiments, the disclosure relates to the combined use ofgrowth factor(s) and compounds disclosed herein such as2,4-diamino-6-vinyl-1,3,5-triazine, Compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazineor derivatives or salts thereof and one or more growth factors in bonegrowth applications. Typically, the growth factor is a bonemorphogenetic proteins (BMPs), including but not limited to, BMP-1,BMP-2, BMP-2A, BMP-2B, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7 (OP-1),BMP-8, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15.BMPs act through specific transmembrane receptors located on cellsurface of the target cells.

Non-limiting examples of additional suitable growth factors includeosteogenin, insulin-like growth factor (IGF)-1, IGF-II, TGF-beta1,TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, osteoinductive factor (OIF),basic fibroblast growth factor (bFGF), acidic fibroblast growth factor(aFGF), platelet-derived growth factor (PDGF), vascular endothelialgrowth factor (VEGF), growth hormone (GH), growth and differentiationfactors (GDF)-5 through 9, and osteogenic protein-1 (OP-1). The growthfactors may be isolated from synthetic methods, recombinant sources ormay be purified from a biological sample. Preferably the growth factorsare obtained from a recombinant technology and for clarity certainembodiments include rhBMP-2, rhBMP-4, rhBMP-6, rhBMP-7, and rhGDF-5, asdisclosed, for example, in the U.S. Pat. Nos. 4,877,864; 5,013,649;5,661,007; 5,688,678; 6,177,406; 6,432,919; 6,534,268, and 6,858,431,and in Wozney, J. M., et al. (1988) Science, 242(4885):1528-1534 herebyincorporated by reference.

In a typical embodiment, a graft composition comprises a matrix, BMP-2,and a compound disclosed herein such as2,4-diamino-6-vinyl-1,3,5-triazine,4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine, a derivative orsalt thereof or combinations of other growth factors such as GDF-5. Inone embodiment, the matrix contains an effective amount of a BMP-2protein, an rhBMP-2 protein, functional fragments thereof, orcombinations thereof. For certain embodiments, the range ofconcentrations of BMP-2 may be about 1.0 to 4.0 mg/ml and GDF-5concentrations may be 0.25 to 4.0 mg/ml. Although a graft matrix may beloaded during manufacturing, it is typically loaded just prior toimplantation.

The transcription of human BMP-2 is 396 amino acids in length, localizedto chromosome 20p12. BMP-2 belongs to the transforming growthfactor-beta (TGF-beta) superfamily. The human amino acid sequence BMP-2is SEQ ID NO:1 shown below. Amino acids 38-268 are the TGF-betapropeptide domain, and 291-396 are the TGF-beta family N-terminaldomain. Amino acids 283-396 are the mature peptide. The mature form ofBMP-2 contains four potential N-linked glycosylation sites perpolypeptide chain, and four potential disulfide bridges. (SEQ ID NO: 1)1 MVAGTRCLLA LLLPQVLLGG AAGLVPELGR RKFAAASSGR PSSQPSDEVL SEFELRLLSM 61FGLKQRPTPS RDAVVPPYML DLYRRHSGQP GSPAPDHRLE RAASRANTVR SFHHEESLEE 121LPETSGKTTR RFFFNLSSIP TEEFITSAEL QVFREQMQDA LGNNSSFHHR INIYEIIKPA 181TANSKFPVTR LLDTRLVNQN ASRWESFDVT PAVMRWTAQG HANHGFVVEV AHLEEKQGVS 241KRHVRISRSL HQDEHSWSQI RPLLVTFGHD GKGHPLHKRE KRQAKHKQRK RLKSSCKRHP 301LYVDFSDVGW NDWIVAPPGY HAFYCHGECP FPLADHLNST NHAIVQTLVN SVNSKIPKAC 361CVPTELSAIS MLYLDENEKV VLKNYQDMVV EGCGCR.

In one embodiment, bone morphogenetic protein includes one of thefollowing synthetic peptide fragments of BMP-2: SEQ ID NO: 2(KIPKASSVPTELSAISTLYLDDD), SEQ ID NO: 3 (CCCCDDDSKIPKASSVPTELSAISTLYL)SEQ ID NO: 4 (C₁₆H₃₁O—NH—CCCCGGGSKIPKASSVPTELSAISTLYL) which may besynthesized by FMOC/tBu solid-phase peptide synthesis.

BMP-7 also belongs to the TGF-beta superfamily. It induces cartilage andbone formation. The amino acid sequence of BMP-7 is SEQ ID NO: 5. (SEQID NO: 5) 1 MHVRSLRAAA PHSFVALWAP LFLLRSALAD FSLDNEVHSS FIHRRLRSQERREMQREILS 61 ILGLPHRPRP HLQGKHNSAP MFMLDLYNAM AVEEGGGPGG QGFSYPYKAVFSTQGPPLAS 121 LQDSHFLTDA DMVMSFVNLV EHDKEFFHPR YHHREFRFDL SKIPEGEAVTAAEFRIYKDY 181 IRERFDNETF RISVYQVLQE HLGRESDLFL LDSRTLWASE EGWLVFDITATSNHWVVNPR 241 HNLGLQLSVE TLDGQSINPK LAGLIGRHGP QNKQPFMVAF FKATEVHFRSIRSTGSKQRS 301 QNRSKTPKNQ EALRMANVAE NSSSDQRQAC KKHELYVSFR DLGWQDWIIAPEGYAAYYCE 361 GECAFPLNSY MNATNHAIVQ TLVHFINPET VPKPCCAPTQ LNAISVLYFDDSSNVILKKY 421 RNNVVRACGC H. Amino acids 1-29 are a potential signalsequence; 30-431 are the prepropeptide, and 293-431 are the matureprotein. The mature form of BMP-7 contains four potential N-linkedglycosylation sites per polypeptide chain, and four potential disulfidebridges.

Graft Compositions

In some embodiments, the disclosure relates to a graft compositioncomprising growth factor(s) and 2,4-diamino-6-vinyl-1,3,5-triazine,Compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazineor derivatives or salts thereof. In some embodiments, these compositionscan be created from polymers, bone granules, and ceramics such ascalcium phosphates (e.g. hydroxyapatite and tricalcium phosphate),bioglass, and calcium sulphate.

Bioglass refers to materials of SiO₂, Na₂O, CaO, and P₂O₅ in specificproportions. The proportions differ from the traditional soda-limeglasses in lower amounts of silica (typically less than 60 mol %),higher amounts of sodium and calcium, and higher calcium/phosphorusratio. A high ratio of calcium to phosphorus promotes formation ofapatite crystals; calcium and silica ions can act as crystallizationnuclei. Some formulations bind to soft tissues and bone, some only tobone, some do not form a bond at all and after implantation getencapsulated with non-adhering fibrous tissue, and others are completelyabsorbed overtime. Mixtures of 35-60 mol % SiO₂, 10-50 mol % CaO, and5-40 mol % Na₂O bond to bone and some formulations bond to soft tissues.Mixtures of >50 mol % SiO₂, <10 mol % CaO, <35 mol % Na₂O typicallyintigrate within a month. Some CaO may be replaced with MgO and someNa₂O may be replaced with K₂O. Some CaO can be replaced with CaF₂.

In some embodiments, the disclosure relates to a graft compositioncomprising growth factor(s) and compounds disclosed herein such as2,4-diamino-6-vinyl-1,3,5-triazine, Compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazineor derivatives or salts thereof and/or polysaccharides such ashyaluronate, cellulose or cellulose derivatives such as, but not limitedto, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, andcarboxymethyl cellulose. Typically, cellulose derivates are used ingraft compositions that produce a paste or putty.

In some embodiments, the disclosure relates to a bone graft compositioncomprising a bone morphogenetic protein and2,4-diamino-6-vinyl-1,3,5-triazine, Compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,derivative, or salt thereof and a graft matrix. The matrix is typicallya polymer designed to hold bone compatible salts, such as calciumphosphates, for replacement during bone growth. An example is a bovineType I collagen embedded with biphasic calcium phosphate granules.Optionally, matrix compositions may also include one or more agents thatsupport the formation, development and growth of new bone, and/or theremodeling thereof. Typical examples of compounds that function in, sucha supportive manner include extracellular matrix-associated boneproteins such as alkaline phosphatase, osteocalcin, bone sialoprotein(BSP) and osteocalcin, phosphoprotein (SPP)-1, type I collagen,fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, andosteopontin.

In certain embodiments, the graft matrix can be made up of a hydrogelpolymer. Typically, a hydrogel is made-up of acrylate polymers andcopolymers substituted with an abundance of hydrophilic groups, such asterminal hydroxy or carboxyl groups. In certain embodiments, the graftcomposition is biodegradable. In certain embodiments, the matrixcomprises homopolymers and copolymers consisting of gylcolide andlactide. For certain embodiments, the graft composition comprises amatrix of hydroxyethylmethacrylate or hydroxymethylmethyacrylatepolymers containing hydroxyapatite in a mineral content approximatelythat of human bone. Such a composition may also be made withcrosslinkers comprising an ester, anhydride, orthoester, amide, orpeptide bond. In some embodiments, crosslinkers contain the followingpolymers: polyethylene glycol (PEG), polylactic acid, polyglycolide orcombinations thereof.

In certain embodiments, the graft composition may contain one or moreantibiotics and/or anti-inflammatory agents. Suitable antibioticsinclude, without limitation, nitroimidazole antibiotics, tetracyclines,penicillins, cephalosporins, carbopenems, aminoglycosides, macrolideantibiotics, lincosamide antibiotics, 4-quinolones, rifamycins andnitrofurantoin. Suitable specific compounds include, without limitation,ampicillin, amoxicillin, benzylpenicillin, phenoxymethylpenicillin,bacampicillin, pivampicillin, carbenicillin, cloxacillin, cyclacillin,dicloxacillin, methicillin, oxacillin, piperacillin, ticarcillin,flucloxacillin, cefuroxime, cefetamet, cefetrame, cefixine, cefoxitin,ceftazidime, ceftizoxime, latamoxef, cefoperazone, ceftriaxone,cefsulodin, cefotaxime, cephalexin, cefaclor, cefadroxil, cefalothin,cefazolin, cefpodoxime, ceftibuten, aztreonam, tigemonam, erythromycin,dirithromycin, roxithromycin, azithromycin, clarithromycin, clindamycin,paldimycin, lincomycirl, vancomycin, spectinomycin, tobramycin,paromomycin, metronidazole, tinidazole, ornidazole, amifloxacin,cinoxacin, ciprofloxacin, difloxacin, enoxacin, fleroxacin, norfloxacin,ofloxacin, temafloxacin, doxycycline, minocycline, tetracycline,chlortetracycline, oxytetracycline, methacycline, rolitetracyclin,nitrofurantoin, nalidixic acid, gentamicin, rifampicin, amikacin,netilmicin, imipenem, cilastatin, chloramphenicol, furazolidone,nifuroxazide, sulfadiazin, sulfametoxazol, bismuth sub salicylate,colloidal bismuth subcitrate, gramicidin, mecillinam, cloxiquine,chlorhexidine, dichlorobenzylalcohol, methyl-2-pentylphenol or anycombination thereof.

Suitable anti-inflammatory compounds include both steroidal andnon-steroidal structures. Suitable non-limiting examples of steroidalanti-inflammatory compounds are corticosteroids such as hydrocortisone,cortisol, hydroxyltriamcinolone, alpha-methyl dexamethasone,dexamethasone-phosphate, beclomethasone dipropionates, clobetasolvalerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolonevalerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortinebutylesters, fluocortolone, fluprednidene (fluprednylidene) acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, cortisone,cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,fluradrenolone, fludrocortisone, diflurosone diacetate, fluocinolone,fluradrenolone acetonide, medrysone, amcinafel, amcinafide,betamethasone and the balance of its esters, chloroprednisone,chlorprednisone acetate, clocortelone, clescinolone, dichlorisone,diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone,fluprednisolone, hydrocorti sone valerate, hydrocortisonecyclopentylpropionate, hydrocortamate, meprednisone, paramethasone,prednisolone, prednisone, beclomethasone dipropionate, triamcinolone.Mixtures of the above steroidal anti-inflammatory compounds may also beused.

Non-limiting examples of non-steroidal anti-inflammatory compoundsinclude nabumetone, celecoxib, etodolac, nimesulide, apasone, gold,oxicams, such as piroxicam, isoxicam, meloxicam, tenoxicam, sudoxicam,the salicylates, such as aspirin, disalcid, benorylate, trilisate,safapryn, solprin, diflunisal, and fendosal; the acetic acidderivatives, such as diclofenac, fenclofenac, indomethacin, sulindac,tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin,fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; thefenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, andtolfenamic acids; the propionic acid derivatives, such as ibuprofen,naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen,indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,tioxaprofen, suprofen, alminoprofen, and tiaprofenic; and the pyrazoles,such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, andtrimethazone.

Bone Grafting Methods

Bone grafting is possible because bone tissue, unlike most othertissues, has the ability to regenerate if provided the space into whichto grow with appropriate chemical signals. With regard to syntheticgrafts, as native bone grows, it typically replaces most or all of theartificial graft material, resulting in an integrated region of newbone. However, with regard to certain embodiments of the disclosure, itis not intended that new bone must remove all artificial material. Inaddition, with regard to certain embodiments of the disclosure, it isnot intended that graft location need contact any other bone of theskeletal system.

In certain embodiments, the disclosure relates to a method of formingbone comprising implanting a graft composition comprising a compounddisclosed herein such as 2,4-diamino-6-vinyl-1,3,5-triazine, Compound12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,derivatives, or salts thereof, in a subject. In certain embodiments, thedisclosure relates to methods of forming bone comprising implanting agraft composition comprising a bone morphogenetic protein andcompound(s) disclosed herein, such as2,4-diamino-6-vinyl-1,3,5-triazine, Compound 12X:1-(4-chlorophenyl)-4,6-diamino-1,2-dihydro-2,2-dimethyl-1,3,5-triazine,derivatives, or salts thereof, in a subject. The graft may be the resultof a void created by surgical removal or created as a result of anattempt to correct a physical abnormality of a bone, such as but notlimited to, cranial bones; frontal, parietal, temporal, occipital,sphenoid, ethmoid; facial bones; mandible, maxilla, palatine, zygomatic,nasal, lacrimal, vomer, inferior nasal conchae; shoulder girdle; scapulaor shoulder blade, clavicle or collarbone; in the thorax; sternum,manubrium, gladiolus, and xiphoid process, ribs; in the vertebralcolumn; cervical vertebrae, thoracic vertebrae; lumbar vertebrae; in thearms, humerus, radius, ulna; in the pelvis; coccyx; sacrum, hip bone(innominate bone or coxal bone); in the legs; femur, patella, tibia, andfibula. It is contemplated that the graft may be added for cosmeticpurposes, e.g., cheek augmentation. In the case of a broken bone orremoval of a bone during surgery, it may be desirable to secure movementof bone structure with a fixation system and remove the system afterbone forms in the implanted graft.

With regard to prostheses, it may be desirable to grow bone betweenexisting bone and an implanted device, or in preparation of an implanteddevice, such as in the case of a hip replacement, knee replacement, anddental implant, i.e., artificial tooth root used to support restorationsthat resemble a tooth or group of teeth.

In some embodiments, the disclosure relates to three-dimensionalstructures made of biocompatible and biodegradable bone graft materialsin the shape of the bone infused with compositions disclosed herein topromote bone growth. Implants can be used to support a number ofprostheses. A typical implant consists of a titanium device. In certainembodiments, the graft compositions disclosed herein contain implants.

With regard to a sinus augmentation or alveolar ridge augmentation,surgery may be performed as an outpatient under general anesthesia, oralconscious sedation, nitrous oxide sedation, intravenous sedation orunder local anesthesia. Bone grafting is used in cases where there is alack of adequate maxillary or mandibular bone in terms of depth orthickness. Sufficient bone is needed in three dimensions to securelyintegrate with the root-like implant. Improved bone height is importantto assure ample anchorage of the root-like shape of the implant.

In a typical procedure, the clinician creates a large flap of thegingiva or gum to fully expose the bone at the graft site, performs oneor several types of block and onlay grafts in and on existing bone, theninstalls a membrane designed to repel unwanted infection-causingbacteria. Then the mucosa is carefully sutured over the site. Togetherwith a course of systemic antibiotics and topical antibacterial mouthrinses, the graft site is allowed to heal. The bone graft produces livevascular bone and is therefore suitable as a foundation for the dentalimplants.

In certain embodiments, the disclosure relates to methods of performingspinal fusion using compositions disclosed herein. Typically thisprocedure is used to eliminate the pain caused by abnormal motion of thevertebrae by immobilizing the vertebrae themselves. Spinal fusion isoften done in the lumbar region of the spine, but the term is notintended to be limited to method of fusing lumbar vertebrae. Patientsdesiring spinal fusion may have neurological deficits or severe painwhich has not responded to conservative treatment. Conditions wherespinal fusion may be considered include, but are not limited to,degenerative disc disease, spinal disc herniation, discogenic pain,spinal tumor, vertebral fracture, scoliosis, kyphosis (i.e,Scheuermann's disease), spondylolisthesis, or spondylosis.

In certain embodiments, different methods of lumbar spinal fusion may beused in conjunction with each other. In one method, one places the bonegraft between the transverse processes in the back of the spine. Thesevertebrae are fixed in place with screws and/or wire through thepedicles of each vertebra attaching to a metal rod on each side of thevertebrae. In another method, one places the bone graft between thevertebra in the area usually occupied by the intervertebral disc. Inpreparation for the spinal fusion, the disc is removed entirely. Adevice may be placed between the vertebrae to maintain spine alignmentand disc height. The intervertebral device may be made from eitherplastic or titanium or other suitable material. The fusion then occursbetween the endplates of the vertebrae. Using both types of fusion arecontemplated.

Therapeutic Applications

In some embodiments, the disclosure relates to pharmaceuticalcompositions comprising compounds disclosed herein for therapeuticapplications. In some embodiments, the disclosure relates to methods oftreating bone degenerative disorders, such as osteoporosis, osteitisdeformans (“Paget's disease of bone”), bone metastasis (with or withouthypercalcaemia), multiple myeloma, primary hyperparathyroidism, orosteogenesis imperfecta. Osteoporosis is a disease of bones that leadsto an increased risk of fracture. In osteoporosis, the bone mineraldensity (BMD) is reduced, bone microarchitecture is disrupted, and theamount and variety of proteins in bone is altered. Osteoporosis is mostcommon in women after menopause, when it is called postmenopausalosteoporosis, but may also develop in men, and may occur in anyone inthe presence of particular hormonal disorders and other chronic diseasesor as a result of medications, specifically glucocorticoids, when thedisease is called steroid- or glucocorticoid-induced osteoporosis (SIOPor GIOP).

Osteoporotic fractures are those that occur in situations where healthypeople would not normally break a bone; they are therefore regarded asfragility fractures. Typical fragility fractures occur in the vertebralcolumn, rib, hip and wrist. The diagnosis of osteoporosis can be madeusing conventional radiography by measuring the bone mineral density(BMD).

In some embodiments, the disclosure relates to treating bonedegenerative disorders by administering pharmaceutical compositiondescribed herein in combination with other agents, such as calciumcarbonate and calcium citrate, vitamine D, cholecalciferol,1,25-dihydroxycholecalciferol, calcitriol, estrogen, testosterone,raloxifene, pamidronate, neridronate, olpadronate, alendronate(Fosamax), ibandronate (Boniva), risedronate (Actonel), zoledronate(Zometa, Aclasta), etidronate (Didronel), clodronate (Bonefos, Loron),or tiludronate (Skelid).

Formulations

Pharmaceutical compositions disclosed herein may be in the form ofpharmaceutically acceptable salts, as generally described below. Somepreferred, but non-limiting examples of suitable pharmaceuticallyacceptable organic and/or inorganic acids are hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citricacid, as well as other pharmaceutically acceptable acids known per se(for which reference is made to the references referred to below).

When the compounds of the disclosure contain an acidic group as well asa basic group, the compounds of the disclosure may also form internalsalts, and such compounds are within the scope of the disclosure. Whenthe compounds of the disclosure contain a hydrogen-donating heteroatom(e.g. NH), the disclosure also covers salts and/or isomers formed bytransfer of said hydrogen atom to a basic group or atom within themolecule.

Pharmaceutically acceptable salts of the compounds include the acidaddition and base salts thereof. Suitable acid addition salts are formedfrom acids which form non-toxic salts. Examples include the acetate,adipate, aspartate, benzoate, besylate, bicarbonate/carbonate,bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate,esylate, formate, fumarate, gluceptate, gluconate, glucuronate,hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate,saccharate, stearate, succinate, tannate, tartrate, tosylate,trifluoroacetate and xinofoate salts. Suitable base salts are formedfrom bases which form non-toxic salts. Examples include the aluminium,arginine, benzathine, calcium, choline, diethylamine, diolamine,glycine, lysine, magnesium, meglumine, olamine, potassium, sodium,tromethamine and zinc salts. Hemisalts of acids and bases may also beformed, for example, hemisulphate and hemicalcium salts. For a review onsuitable salts, see Handbook of Pharmaceutical Salts: Properties,Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporatedherein by reference.

The compounds described herein may be administered in the form ofprodrugs. A prodrug can include a covalently bonded carrier whichreleases the active parent drug when administered to a mammaliansubject. Prodrugs can be prepared by modifying functional groups presentin the compounds in such a way that the modifications are cleaved,either in routine manipulation or in vivo, to the parent compounds.Prodrugs include, for example, compounds wherein a hydroxy group isbonded to any group that, when administered to a mammalian subject,cleaves to form a free hydroxy group. Examples of prodrugs include, butare not limited to, acetate, formate and benzoate derivatives of alcoholfunctional groups in the compounds. Methods of structuring a compound asprodrugs can be found in the book of Testa and Mayer, Hydrolysis in Drugand Prodrug Metabolism, Wiley (2006). Typical prodrugs form the activemetabolite by transformation of the prodrug by hydrolytic enzymes, thehydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxidesor the cleavage of esters of inorganic acids.

Pharmaceutical compositions for use in the present disclosure typicallycomprise an effective amount of a compound and a suitable pharmaceuticalacceptable carrier. The preparations may be prepared in a manner knownper se, which usually involves mixing the at least one compoundaccording to the disclosure with the one or more pharmaceuticallyacceptable carriers, and, if desired, in combination with otherpharmaceutical active compounds, when necessary under asepticconditions. Reference is made to U.S. Pat. No. 6,372,778, U.S. Pat. No.6,369,086, U.S. Pat. No. 6,369,087 and U.S. Pat. No. 6,372,733 and thefurther references mentioned above, as well as to the standardhandbooks, such as the latest edition of Remington's PharmaceuticalSciences.

Generally, for pharmaceutical use, the compounds may be formulated as apharmaceutical preparation comprising at least one compound and at leastone pharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activecompounds.

The pharmaceutical preparations of the disclosure are preferably in aunit dosage form, and may be suitably packaged, for example in a box,blister, vial, bottle, sachet, ampoule or in any other suitablesingle-dose or multi-dose holder or container (which may be properlylabeled); optionally with one or more leaflets containing productinformation and/or instructions for use. Generally, such unit dosageswill contain between 1 and 1000 mg, and usually between 5 and 500 mg, ofthe at least one compound of the disclosure, e.g. about 10, 25, 50, 100,200, 300 or 400 mg per unit dosage.

The compounds can be administered by a variety of routes including theoral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. The compound will generally be administered in an“effective amount”, by which is meant any amount of a compound that,upon suitable administration, is sufficient to achieve the desiredtherapeutic or prophylactic effect in the subject to which it isadministered. Usually, depending on the condition to be prevented ortreated and the route of administration, such an effective amount willusually be between 0.01 to 1000 mg per kilogram body weight of thepatient per day, more often between 0.1 and 500 mg, such as between 1and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg,per kilogram body weight of the patient per day, which may beadministered as a single daily dose, divided over one or more dailydoses. The amount(s) to be administered, the route of administration andthe further treatment regimen may be determined by the treatingclinician, depending on factors such as the age, gender and generalcondition of the patient and the nature and severity of thedisease/symptoms to be treated. Reference is again made to U.S. Pat. No.6,372,778, U.S. Pat. No. 6,369,086, U.S. Pat. No. 6,369,087 and U.S.Pat. No. 6,372,733 and the further references mentioned above, as wellas to the standard handbooks, such as the latest edition of Remington'sPharmaceutical Sciences.

For an oral administration form, the compound can be mixed with suitableadditives, such as excipients, stabilizers or inert diluents, andbrought by means of the customary methods into the suitableadministration forms, such as tablets, coated tablets, hard capsules,aqueous, alcoholic, or oily solutions. Examples of suitable inertcarriers are gum arabic, magnesia, magnesium carbonate, potassiumphosphate, lactose, glucose, or starch, in particular, corn starch. Inthis case, the preparation can be carried out both as dry and as moistgranules. Suitable oily excipients or solvents are vegetable or animaloils, such as sunflower oil or cod liver oil. Suitable solvents foraqueous or alcoholic solutions are water, ethanol, sugar solutions, ormixtures thereof. Polyethylene glycols and polypropylene glycols arealso useful as further auxiliaries for other administration forms. Asimmediate release tablets, these compositions may containmicrocrystalline cellulose, dicalcium phosphate, starch, magnesiumstearate and lactose and/or other excipients, binders, extenders,disintegrants, diluents and lubricants known in the art.

When administered by nasal aerosol or inhalation, the compositions maybe prepared according to techniques well-known in the art ofpharmaceutical formulation and may be prepared as solutions in saline,employing benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, and/or othersolubilizing or dispersing agents known in the art. Suitablepharmaceutical formulations for administration in the form of aerosolsor sprays are, for example, solutions, suspensions or emulsions of thecompounds of the disclosure or their physiologically tolerable salts ina pharmaceutically acceptable solvent, such as ethanol or water, or amixture of such solvents. If required, the formulation can alsoadditionally contain other pharmaceutical auxiliaries such assurfactants, emulsifiers and stabilizers as well as a propellant.

For subcutaneous or intravenous administration, the compounds, ifdesired with the substances customary therefore such as solubilizers,emulsifiers or further auxiliaries are brought into solution,suspension, or emulsion. The compounds of formula I can also belyophilized and the lyophilizates obtained used, for example, for theproduction of injection or infusion preparations. Suitable solvents are,for example, water, physiological saline solution or alcohols, e.g.ethanol, propanol, glycerol, sugar solutions such as glucose or mannitolsolutions, or mixtures of the various solvents mentioned. The injectablesolutions or suspensions may be formulated according to known art, usingsuitable non-toxic, parenterally-acceptable diluents or solvents, suchas mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodiumchloride solution, or suitable dispersing or wetting and suspendingagents, such as sterile, bland, fixed oils, including synthetic mono- ordiglycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, theformulations may be prepared by mixing the compounds of formula I with asuitable non-irritating excipient, such as cocoa butter, syntheticglyceride esters or polyethylene glycols, which are solid at ordinarytemperatures, but liquefy and/or dissolve in the rectal cavity torelease the drug.

In certain embodiments, it is contemplated that these compositions canbe extended release formulations. Typical extended release formationsutilize an enteric coating. Typically, a barrier is applied to oralmedication that controls the location in the digestive system where itis absorbed. Enteric coatings prevent release of medication before itreaches the small intestine. Enteric coatings may contain polymers ofpolysaccharides, such as maltodextrin, xanthan, scleroglucan dextran,starch, alginates, pullulan, hyaloronic acid, chitin, chitosan and thelike; other natural polymers, such as proteins (albumin, gelatin etc.),poly-L-lysine; sodium poly(acrylic acid);poly(hydroxyalkylmethacrylates) (for examplepoly(hydroxyethylmethacrylate)); carboxypolymethylene (for exampleCarbopol™); carbomer; polyvinylpyrrolidone; gums, such as guar gum, gumarabic, gum karaya, gum ghatti, locust bean gum, tamarind gum, gellangum, gum tragacanth, agar, pectin, gluten and the like; poly(vinylalcohol); ethylene vinyl alcohol; polyethylene glycol (PEG); andcellulose ethers, such as hydroxymethylcellulose (HMC),hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC),methylcellulose (MC), ethylcellulose (EC), carboxyethylcellulose (CEC),ethylhydroxyethylcellulose (EHEC), carboxymethylhydroxyethylcellulose(CMHEC), hydroxypropylmethyl-cellulose (HPMC),hydroxypropylethylcellulose (HPEC) and sodium carboxymethylcellulose (NaCMC); as well as copolymers and/or (simple) mixtures of any of the abovepolymers. Certain of the above-mentioned polymers may further becrosslinked by way of standard techniques.

The choice of polymer will be determined by the nature of the activeingredient/drug that is employed in the composition of the disclosure aswell as the desired rate of release. In particular, it will beappreciated by the skilled person, for example in the case of HPMC, thata higher molecular weight will, in general, provide a slower rate ofrelease of drug from the composition. Furthermore, in the case of HPMC,different degrees of substitution of methoxyl groups and hydroxypropoxylgroups will give rise to changes in the rate of release of drug from thecomposition. In this respect, and as stated above, it may be desirableto provide compositions of the disclosure in the form of coatings inwhich the polymer carrier is provided by way of a blend of two or morepolymers of, for example, different molecular weights in order toproduce a particular required or desired release profile.

Microspheres of polylactide, polyglycolide, and their copolymerspoly(lactide-co-glycolide) may be used to form sustained-release proteindelivery systems. Proteins can be entrapped in thepoly(lactide-co-glycolide) microsphere depot by a number of methods,including formation of a water-in-oil emulsion with water-borne proteinand organic solvent-borne polymer (emulsion method), formation of asolid-in-oil suspension with solid protein dispersed in a solvent-basedpolymer solution (suspension method), or by dissolving the protein in asolvent-based polymer solution (dissolution method). One can attachpoly(ethylene glycol) to proteins (PEGylation) to increase the in vivohalf-life of circulating therapeutic proteins and decrease the chance ofan immune response.

Experimental Compound Screenings

Compounds were selected by virtual screening for development based oncomputational modeling, docking, and in silico screening. It wasdesirable for the compounds to possess features similar to the PPXYmotif of Smads (natural target proteins) that interact with Smurf1, asshown by Ludi3 docking scores. (See Sangadala et al. (2007) Proteins 68:690-701 and FIG. 2). The BMP-potentiating activities of compounds may beevaluated by monitoring several markers of the osteoblastic phenotypecorresponding to various time points during phenotype differentiation ofC2Cl2 cells towards terminally differentiated osteoblasts.

Cell Culture

Mouse C2Cl2 cells and Dulbecco's modified Eagle's medium (DMEM) werepurchased from ATCC (Manassas, Va.). The non-heat inactivated fetalbovine serum (FBS) was purchased from HyClone Laboratories, Inc. (Logan,Utah). The C2Cl2 cells at passages 5 to 10 were subcultured in T-75 cm²flasks in DMEM supplemented with 10% FBS at 37° C. in 5% CO2 withhumidification. When the flasks reached 80% confluence, the cells weretrypsinized and seeded in triplicate at 200,000 cells/well in a 6-wellplate for quantitative real-time RT-PCR and alkaline phosphatase (ALP)assays or at 50,000 cells/well in a 12-well plate for thedual-luciferase reporter assay.

RNA Extraction and Reverse Transcription

The C2Cl2 cells were plated at a density of 200,000 cells/well in 6-wellplates and grown overnight in DMEM containing 10% FBS. On day 2, theculture medium was replaced with DMEM containing 2% FBS and the cellswere treated with various concentrations of Smurf1-interacting compound(diluted from 10 mg/ml stock solutions prepared in DMSO) for 24 hours.In control cultures, a DMSO solvent concentration of 0.01% (v/v) wasapplied. On day 3, medium was replaced with fresh DMEM containing 2% FBSand the cells were treated with BMP-2 for 24 hours.

Total RNA was harvested using the RNeasy Mini Kit according to themanufacturer's instructions (Qiagen, Valencia, Calif.). The harvestedRNA was digested with RNase-free DNase I (Qiagen, Valencia, Calif.) toremove DNA contamination. The concentration of the isolated RNA wasdetermined by measuring the absorbance at 260 nm wave length with aspectrophotometer (Model DU 640, Beckman Coulter, Inc. Brea, Calif.).The ratio of A260/A280 was between 1.6 and 1.8. Reverse transcriptionwas carried out to synthesize cDNA in a 100 μl volume with 2 μg of totalRNA, 10×RT buffer, 5.5 mM MgCl2, 2 mM dNTP mixture, 0.125 μM oligo d(T),0.125 μM random primer, 40 Units of RNase inhibitor, and 125 Units ofMultiScribe (Applied Biosystems, Foster City, Calif.) for 10 minutes at25° C., 30 minutes at 48° C., and 5 minutes at 95° C.

Quantitative Real-Time RT-PCR

Quantitative real-time RT-PCR was performed to determine the mRNAexpression level of ALP and osteocalcin. The sequences of the primerswere as follows: ALP (forward, 5′-TCA GGG CAA TGA GGT CAC ATC-3′; (SEQID NO: 6), reverse, 5′-CAC AAT GCC CAC GGA CTT C-3′) (SEQ ID NO: 7),osteocalcin (forward, 5′-CGG CCC TGA GTC TGA CAA AG-3′; (SEQ ID NO: 8)reverse, 5′-CTC GTC ACA AGC AGG GTC AA-3′ (SEQ ID NO: 9)). Twenty-fivemicroliters of reaction volume included 5μl of cDNA, 0.5 μl of 10 μM ofeach primer and 12.5 μl of 2×SYBR green master mix (Applied Biosystems).Real-time PCR was performed with the following three-step protocol: step1, 50° C. for 2 minutes; step 2, 95° C. for 10 minutes; step 3, 40cycles of 95° C. for 15 seconds and 62° C. for 1 minute using the 7500real-time PCR System (Applied Biosystems, Foster City, Calif.). Toconfirm the amplification specificity, the PCR products were subjectedto a dissociation curve analysis. The threshold cycles (Ct) of eachreaction were normalized to those obtained for 18S mRNA using the −ΔΔCtmethod (Applied Biosystems). All PCR reactions were performed intriplicate.

Alkaline Phosphatase (ALP) Assay

The C2Cl2 cells were plated at 200,000 cells/well in 6-well plates andgrown overnight in DMEM containing 10% FBS. On day 2, the culture mediumwas replaced with DMEM containing 2% FBS and the cells were treated withvarious concentrations of the Smurf1-interacting compound for 24 hours.On day 3, the medium was replaced with fresh DMEM containing 2% FBS andthe cells were treated with 50 ng/ml of BMP-2 with or without compoundfor 72 hours. The cells were washed with phosphate-buffered saline (PBS)and lysed by addition of lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM MgCl2and 0.5% Triton X-100). The cell lysates were centrifuged for 5 minutesat 13,000×g. The supernatant was removed and the aliquots were assayedfor ALP activity and protein amount. The ALP activity was measured intriplicate using an ALP assay kit (Sigma-Aldrich, St. Louis, Mo.) inmicrotiter plates. The protein amount was determined with Bio-Radprotein assay reagent (Bio-Rad, Hercules, Calif.) using bovine serumalbumin (BSA) as a standard. The ALP activity (nmoles of p-nitrophenolper ml) was normalized to the protein amount (nmoles of p-nitrophenolper μg).

Dual-Luciferase Reporter Assay

A BMP-specific Smad1-driven 9×GCCG (a consensus binding sequence forSmad1) reporter plasmid was used. The C2Cl2 cells were trypsinized andseeded in triplicate wells at 50,000 cells/well in 12-well plates onday 1. On day 2, the cells were cotransfected with the 9×GCCG-reporterconstruct and the renilla-luciferase control vector using SuperFect(Qiagen, Valencia, Calif.) for 24 hrs. A total of 1 μg of plasmids wasused for cotransfection in each well, and the concentration ofrenilla-luciferase vector was 1/15 of the 9×GCCG-reporter plasmid. Onday 3, medium was replaced with DMEM containing 2% FBS and the cellswere treated with various concentrations of the Smurf1-interactingcompound. On day 4, the cells were treated with BMP-2. On day 5, theluciferase activities were measured in 20 μl of cell-lysate using thedual-luciferase assay system (Promega, Madison, Wis.) with a luminometer(LumiCount; Packard Bioscience, Meriden, Conn.) following themanufacturer's instructions. The luciferase activity was expressed asrelative units of luciferase (RUL; a ratio of firefly luciferase torenilla luciferase activity).

Determination of EC₅₀

The EC₅₀ values were calculated by determining the concentration bywhich 50% of maximum activity was reached using the sigmoidal fitequation. The 50% effective concentrations were determined with thestandard curve analysis of SigmaPlot 8.02. The nonlinear regressionequation is y=min+(max-min)/(1+(x/EC₅₀)Hill Slope) where y is theobserved responses; x is the dose concentration; max and min areapproximated by the program automatically during the calculation. Valueswere not extrapolated beyond the tested range of concentrations.

1. A bone graft composition comprising a1-(phenyl)-5-isopropyl-biguanide or salt thereof optionally substitutedwith one or more substituents.
 2. The bone graft composition of claim 1,wherein 1-(phenyl)-5-isopropyl-biguanide optionally substituted with oneor more substituents has formula II:

or a salt thereof wherein R¹ is hydrogen or alkyl; R² is hydrogen oralkyl; R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, or heterocyclyloptionally substituted with one or more the same or different R⁴; R⁴ isalkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, or heterocyclyl,wherein R⁴ is optionally substituted with one or more, the same ordifferent, R⁵; and R⁵ is halogen, nitro, cyano, hydroxy,trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl,mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,methylamino, ethylamino, dimethylamino, diethylamino,N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl,N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl,methylthio, ethylthio, methyl sulfinyl, ethyl sulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
 3. Thebone graft composition of claim 1, wherein the1-(phenyl)-5-isopropyl-biguanide substituted with one or moresubstituents is 1-(4-chlorophenyl)-5-isopropyl-biguanide.
 4. The bonegraft composition of claim 1, further comprising a growth factor,wherein the growth factor is a bone morphogenetic protein.
 5. The bonegraft composition of claim 4, wherein the bone morphogenetic protein isBMP-2, BMP-7, BMP-6, or BMP-9.
 6. The bone graft composition of claim 1,further comprising calcium phosphates.
 7. The bone graft composition ofclaim 6, wherein said calcium phosphates are hydroxyapatite andtricalcium phosphate.
 8. The bone graft composition of claim 1, furthercomprising collagen.
 9. The bone graft composition of claim 1, furthercomprising a hydrogel matrix.
 10. A kit comprising a1-(phenyl)-5-isopropyl-biguanide or salt thereof optionally substitutedwith one or more substituents and a graft composition.
 11. The kit ofclaim 10, wherein 1-(phenyl)-5-isopropyl-biguanide optionallysubstituted with one or more substituents has formula II:

or a salt thereof wherein R¹ is hydrogen or alkyl; R² is hydrogen oralkyl; R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, or heterocyclyloptionally substituted with one or more the same or different R⁴; R⁴ isalkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, or heterocyclyl,wherein R⁴ is optionally substituted with one or more, the same ordifferent, R⁵; and R⁵ is halogen, nitro, cyano, hydroxy,trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl,mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,methylamino, ethylamino, dimethylamino, diethylamino,N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl,N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl,methylthio, ethylthio, methyl sulfinyl, ethyl sulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
 12. Thekit of claim 10, wherein the 1-(phenyl)-5-isopropyl-biguanidesubstituted with one or more sub stituents is1-(4-chlorophenyl)-5-isopropyl-biguanide.
 13. The kit of claim 10,further comprising a growth factor, wherein the growth factor is a bonemorphogenetic protein.
 14. The kit of claim 13, wherein the bonemorphogenetic protein is BMP-2, BMP-7, BMP-6, or BMP-9.
 15. The kit ofclaim 10, wherein the graph composition comprises collagen.
 16. The kitof claim 10, wherein the graph composition comprises a hydrogel matrix.17. A method of forming bone or cartilage, comprising implanting a graftcomposition comprising 1-(phenyl)-5-isopropyl-biguanide or salt thereofoptionally substituted with one or more substituents in a subject underconditions such that bone or cartilage forms in the graft composition.18. The method of claim 17, wherein 1-(phenyl)-5-isopropyl-biguanideoptionally substituted with one or more substituents has formula

or a salt thereof wherein R¹ is hydrogen or alkyl; R² is hydrogen oralkyl; R³ is alkyl, phenyl, benzyl, carbocyclyl, aryl, or heterocyclyloptionally substituted with one or more the same or different R⁴; R⁴ isalkyl, halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy,carbamoyl, alkoxy, alkylthio, alkylamino, (alkyl)₂amino, alkylsulfinyl,alkyl sulfonyl, aryl sulfonyl, carbocyclyl, aryl, or heterocyclyl,wherein R⁴ is optionally substituted with one or more, the same ordifferent, R⁵; and R⁵ is halogen, nitro, cyano, hydroxy,trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy, carbamoyl,mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl, acetoxy,methylamino, ethylamino, dimethylamino, diethylamino,N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl,N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl,methylthio, ethylthio, methyl sulfinyl, ethyl sulfinyl, mesyl, ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.
 19. Themethod of claim 17, wherein the 1-(phenyl)-5-isopropyl-biguanidesubstituted with one or more substituents is1-(4-chlorophenyl)-5-isopropyl-biguanide.
 20. The method of claim 17,wherein the graft composition further comprises a bone morphogeneticprotein.